To develop new biocatalysis processes that are useful in particular to the chemical and pharmaceutical industry and to the engineering of proteins, it is necessary to use methods for detecting reliable, fast, and low-cost catalytic activities.
In the prior art, there are two types of substrates that allow the detection of catalytic activity.
On the one hand, substrates of the type modified by a detectable group that can release a signal after their transformation by a particular catalytic activity, and, on the other hand, the techniques that use natural-type substrates as indicated below are known.
Among the modified substrates, it is possible to use substrates such as aromatic alcohol ester that release, after their transformation, a tinted or fluorescent aromatic alcohol (D. C. Demirjian et al. Top. Curr. Chem. 1999, 200, 1). These substrate types have a major disadvantage, because the chromogenic molecule or fluorogenic molecule is a highly activated group, which makes these substrates unstable. The detection reactions that are derived therefrom consequently can be non-specific.
A second class of modified substrates results in the revelation of a product that is obtained after secondary enzymatic reaction and/or spontaneous reaction (N. Jourdain et al. Tetrahedron Lett. 1998, 39, 9415; K. L. Matta et al., Carbohydr. Res. 1981, 90, C1–C3; G. Klein and J. L. Reymond, Helv. Chim. Acta 1999, 82, 400). This second class of substrates is more stable. This second class of substrates, however, is limited to particular uses. Actually, the dosage of the reaction is done directly on the released product by using an enzyme. The released product should therefore correspond to very specific structural characteristics, which thereby limits the diversity of catalytic activities that can be detected.
A third class of modified substrates has been developed to detect catalytic activities by avoiding the problems raised in advance (Badalassi, F. et al. Angew Chem Int Ed Engl. 2000, 39 (22): 4067–4070). These substrates, however, always correspond to modified substrates.
In contrast, the natural substrates that can demonstrate a catalytic activity are known. The various techniques, however, that use natural substrates are often complex and cumbersome to use for high-flow screening (cumbersome instrumentation in the case of the IR thermography, CE, HPLC, GC MS) or limited to particular catalytic activities that can, for example, induce a difference in pH and/or in the narrowly defined reaction conditions (measurement of pH variations, secondary enzymes). Most of these measurements are also very expensive to carry out because of the cost or the instrumentation, or reagents involved, in particular the secondary enzymes and the anti-product antibodies (M. T. Reetz et al Angew. Chem. 1999, 1111, 1872; A. Holzwarth et al. Angew. Chem. 1998, 110, 2788; M. T. Reetz et al. Angew. Chem. 2000, 112, 1294, Angew. Chem. Int. Ed. Engl. 2000, 39, 1236; Taran et al. Tetrahedron Lett. 1999, 40, 1887, 1891, S. M. Firestine et al. Nat. Biotechnol 2000, 18, 544; F. Moris-Varas et al. Bioorg. Med. Chem. 1999, 7, 2183).